Marcell Cserhalmi

Suppression of innate and adaptive B cell activation pathways by antibody coengagement of FcγRIIb and CD19

The Fc receptor (FcγRIIb) inhibits B cell responses when coengaged with B cell receptor (BCR), and has become a target for new autoimmune disease therapeutics. For example, BCR and FcγRIIb coengagement via the Fc-engineered anti-CD19 XmAb5871 suppresses humoral immune responses. We now assess effects of XmAb5871 on other activation pathways, including the pathogen-associated molecular pattern receptor, TLR9. Since TLR9 signaling is implicated in autoimmune diseases, we asked if XmAb5871 could inhibit TLR9 costimulation. We show that XmAb5871 decreases ERK and AKT activation, cell proliferation, cytokine, and IgG production induced by BCR and/or TLR9 signals. XmAb5871 also inhibited differentiation of citrullinated peptide-specific plasma cells from rheumatoid arthritis patients. XmAb5871 may therefore have potential to suppress pathogenic B cells in autoimmune diseases.

FHR-1 binds to C-reactive protein and enhances rather than inhibits complement activation

Factor H–related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

The Murine Factor H-Related Protein FHR-B Promotes Complement Activation

Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

A1.84 Switching off B cells by Fc-engineered anti-CD19 antibody (XmAb5871)

Background and Objectives CD19 is a B cell-specific marker present on pre-B cells, immature and mature B cells but lacking on plasma cells. CD19 is a positive co-receptor of B cell receptors (BCR) and a target of the tyrosine kinase Lyn in activated B cells. Phosphorylated CD19 recruits Lyn, PI3K and Vav, enhancing B cell activation. In contrast, FcgRIIb is also a target of Lyn, but when co-engaged with BCR is phosphorylated, recruits SHP2 and SHIP phosphatases, and inhibits B cell activation. XmAb5871 is an Fc engineered anti-CD19 that binds to FcgRIIb with approximately 400 higher affinity as compared to its native IgG1 Fc counterpart (Horton HM et al. J. Immunol. 2011, 186:4223-4233). Our aim was to clarify if Fc-engineered CD19 antibody amplifying the FcgRIIb-mediated inhibitory pathway in activated B cells is able to inhibit not only BCR-mediated but also TLR9-stimulated signals. The innate receptor TLR9 plays a role in the etiology of systemic autoimmune diseases by lowering the signalling threshold of B cells. An agent that could inhibit both BCR- and TLR9-mediated signals may block autoantibody production without B cell depletion, and have therapeutic potential in systemic autoimmune diseases. Materials and Methods We studied early and late signalling events in B cells purified from human blood or tonsils by negative selection. The cells were stimulated by single, double or triple combined signals via BCR, TLR9 and CD19. We monitored the phosphorylation of FcgRIIb and ERK, induction of the activation marker CD38, cell proliferation, cytokine and antibody production of B cells. Results We show that XmAb5871 decreases BCR-induced ERK and AKT activation in B cells. In functional assays, we found that cell proliferation, plasma cell differentiation and cytokine production induced by the combined BCR and TLR9-mediated signals were inhibited by XmAb5871. The synergistically enhanced secretion of IL-6, IL-10 and TNFα induced by the BCR and TLR9 double signals was almost completely blocked by XmAb5871. Moreover, the Fc-engineered anti-CD19 inhibited the differentiation of citrullinated filaggrin peptide-specific antibody-forming cells from rheumatoid arthritis patients. Conclusion These data indicate that an Fc-engineered anti-CD19 antibody amplifying FcgRIIb inhibits activated B cells including autoantibody as well as cytokine production, and thus might be a useful new therapeutic strategy to suppress pathogenic B cells in autoimmune disease. Acknowledgement The work was supported by the National Development Agency OTKA 80689 fund.

Functional characterization of the disease-associated N-terminal complement factor H Mutation W198R

Dysregulation of the complement alternative pathway is involved in the pathogenesis of several diseases, including the kidney diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). In a patient, initially diagnosed with chronic glomerulonephritis, possibly C3G, and who 6 years later had an episode of aHUS, a heterozygous missense mutation leading to a tryptophan to arginine exchange (W198R) in the factor H (FH) complement control protein (CCP) 3 domain has previously been identified. The aim of this study was to clarify the functional relevance of this mutation. To this end, wild-type (FH1–4WT) and mutant (FH1–4W198R) CCPs 1–4 of FH were expressed as recombinant proteins. The FH1–4W198R mutant showed decreased C3b binding compared with FH1–4WT. FH1–4W198R had reduced cofactor and decay accelerating activity compared with the wild-type protein. Hemolysis assays demonstrated impaired capacity of FH1–4W198R to protect rabbit erythrocytes from human complement-mediated lysis, and also to prevent lysis of sheep erythrocytes in human serum induced by a monoclonal antibody binding in FH CCP5 domain, compared with that of FH1–4WT. Thus, the FH W198R exchange results in impaired complement alternative pathway regulation. The heterozygous nature of this mutation in the index patient may explain the manifestation of two diseases, likely due to different triggers leading to complement dysregulation in plasma or on cell surfaces.